Exploring urinary modified nucleosides as biomarkers for diabetic retinopathy: Development and validation of a ultra performance liquid chromatography-tandem mass spectrometry method

The dynamic modification of RNA plays a crucial role in biological regulation and is strongly linked to human disease development and progression. Notably, modified nucleosides in urine have shown promising potential as early diagnostic biomarkers for various conditions. In this study, we developed and validated a rapid, sensitive, and accurate UPLC-MS/MS method for quantifying eight types of modified nucleosides ( N 1 -methyladenosine (m 1 A), N 6 -methyladenosine (m 6 A), 5-methyluridine (m 5 U), 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 5-methylcytidine (m 5 C), 2'-O-methylcytidine (Cm), N 1 -methylguanosine (m 1 G), and N 7 -methylguanosine (m 7 G) in human urine. Using the method, we measured the urinary concentrations of m 1 A, m 6 A, m 5 U, τm 5 s 2 U, m 5 C, Cm, m 1 G, and m 7 G in a total of 21 control individuals and 23 patients diagnosed with diabetic retinopathy (DR). Cm levels showed promise as a diagnostic marker for diabetic retinopathy (DR), with a significant value (P < 0.01) and an AUC of 0.735. Other modified nucleosides also exhibited significant differences within specific subpopulations. As non-proliferative diabetic retinopathy (NPDR) signifies the latent early stage of diabetic retinopathy, we developed a multivariate linear model that integrates patients' sex, age, height, and urinary concentration of modified nucleosides which aims to predict and differentiate between healthy individuals, NPDR patients, and proliferative diabetic retinopathy (PDR) patients. Encouragingly, the model achieved satisfactory accuracy rates: healthy (81%), NPDR (75%), and PDR (80%). Our findings provide valuable insights into the development of an early, cost-effective, and noninvasive diagnostic approach for diabetic retinopathy.